Tumor-derived GDF-15 blocks LFA-1 dependent T cell recruitment and suppresses responses to anti-PD-1 treatment

Immune checkpoint blockade therapy is beneficial and even curative for some cancer patients. However, the majority don’t respond to immune therapy. Across different tumor types, pre-existing T cell infiltrates predict response to checkpoint-based immunotherapy. Based on in vitro pharmacological studies, mouse models and analyses of human melanoma patients, we show that the cytokine GDF-15 impairs LFA-1/β2-integrin-mediated adhesion of T cells to activated endothelial cells, which is a pre-requisite of T cell extravasation. In melanoma patients, GDF-15 serum levels strongly correlate with failure of PD-1-based immune checkpoint blockade therapy. Neutralization of GDF-15 improves both T cell trafficking and therapy efficiency in murine tumor models. Thus GDF-15, beside its known role in cancer-related anorexia and cachexia, emerges as a regulator of T cell extravasation into the tumor microenvironment, which provides an even stronger rationale for therapeutic anti-GDF-15 antibody development.

nodes (skin). Adoptive transfer experiments with CFSE labeled CD8 and CD4 populations will allow to track both the homing and initial activation of naïve T cells in TdLNs. Memory polyclonal T cells raised against the tumor in donor mice should be also tested. 11. GDF-15 seem to exert multiple effects on the tumor environment via metabolic changes which are associated with cachexia (Fig. 3a). The authors don't attempt to link this finding to LFA-1 inhibition or to interference with T cell infiltration. This is puzzling and confusing. 12. The use of humanized mice in this study is questionable and should be re-considered since it is a very complex experimental system. I don't see what advantages it has over immunocompetent mice since as noted, among other deficiencies APCs in these mice are scarce or abnormal. It is not well explained how the different T cells emerge in this system, and how they distribute in the spleen and blood. I'd consider removing this model and describing it in another paper. 13. The clinical data and especially the correlations between elevated serum levels of GDF-15 and survival are nice and statistically significant. Nevertheless, in light of the multiple effects of this cytokine it is hard to directly link these results to the main theme of the paper which is LFA-1 inhibition and attenuation of trafficking and function of tumor killer immune cells in the TME. So is the inclusion of data from pregnant women. It is unclear how these data strengthen the main theme of this work. I think it does the very opposite.
Minor comments 1. The terminology 'recruitment' used in the legend of Fig. 1 is confusing. In vitro readouts do not involve recruitment. Please change it. The explanation of these in vitro readouts in the main text should be also improved. 2. The title "GDF-15 acts on naïve and memory CD8+, and on CD4+ memory T cells" must be revisited. As far as I can tell, naïve T cells are less responsive to . The mentioning of "catch bonds" is relevant to the discussion but not to the result section since this biophysical property is not directly measured in this study. 4. Fig. 4a-the intensity of staining (CD45 and CD8) is very different for the different tumors. Please explain. 5. Adjuvant immunization of tumor bearing mice is mentioned in the methods but never discussed in the main text. Why? 6. Why can't alternative adhesive pathways such as VLA-4-VCAM-1 compensate for LFA-1 inhibition in the various models. This point has to be discussed. 7. LFA-1 inhibition by GDF-1 may result in reduced functionality due to poor ability of the CTLs to generate killing synapses with tumor cells? This point should be further emphasized.
Reviewer #2 (Remarks to the Author): In this study, Haake and colleagues have investigated the influence of GDF-15 on T-cell recruitment within the tumor microenvironment and on the response to anti-PD-1 immunotherapy. In the first part of the work, the authors provided in vitro evidence of a role of GDF-15 in interfering with the LFA-1/ICAM1 interaction, which is known to be essential for T-cell migration and extravasation. In a second part, using the murine MC38 colon cancer model, they attempted to functionally validate these observations in vivo, and investigated the consequences of GDF15 overexpression on T-cell infiltration and response/resistance to anti-PD1 therapy. In addition, they assessed the potential of using anti-GDF15 to improve anti-PD-1 effect in this setting. Data confirmed the prognostic value of GDF15, and a trend to a synergistic effect for the anti-PD1/anti-GDF15 combination was observed in this model. In the last part, the authors examined various datasets and cohort of patients (Melanoma and oropharyngeal squamous cell carcinomas) to test the impact of GDF15 expression levels (by IHC and serum measures) on patient clinical outcomes and tumor T-cell infiltration. They show a positive association of GDF-15 with poor survival, and inverse correlation with T-cell infiltrates (CD8, CD3, Treg). They also report evidence in two series of metastatic melanoma patients that elevated GDF-15 serum levels are associated with resistance to PD-1 blockade monotherapy, suggesting GDF-15 as a novel predictive biomarker in this cancer type. The topic is of interest and original. The study provides results of relevance for the oncoimmunology field. However, several technical and conceptual concerns need to be addressed.
Major comments: 1-in the first part of the manuscript, in vitro experiments and associated conclusions derive from T cells purified from healthy donor PBMCs. The significance would be stronger if the authors could expand the work and confirm the findings using T lymphocytes from cancer patients.
2-page 8 and Supp. Figure S2, authors claim that "GDF-15 acts on naïve and memory CD8+, and on CD4+ memory T cells". But it is unclear how they have differentiated the memory subsets from naïve T cells. Information is missing in the method and Figure. It would be important to provide flow cytometry data to support the observed differences and statements (GDF15/anti-LFA1 treatments). Of note, in figure 2, adhesion data are shown for pan-, naïve-, Memory and EM (effector memory) CD4+ T cells. However, for CD8+ cells, the EM category is missing. Moreover, the quality of the images in panels a and b needs to be improved.
3-Data in figure 3 are difficult to interpret. For example, sample size and statistics in panels c, f, g make the current data questionable and hard to interpret (with 2-to-4 points in each group) 4- Fig 3i, j, k, tumor growth curves for each treatment are presented as various sets. It would be keen for the authors to include combined curves to help visualization and interpretation of the added value of these treatments relative to the control condition. Figure 3k and Page 10 (L14), authors claim "Adding anti-GDF-15 to anti-PD-1, however, resulted in complete clearance of MC38hGDF-15 tumors in 4/10 mice (Fig. 3k), indicating that anti-GDF-15 and PD-1-based immune checkpoint blockade synergize to increase survival in mice". This is a key statement but this relies only on 4 events, with no significant differences for survival between the two conditions (p= 0.17). This could be an interesting observation, but parts of the experiments should be repeated to foster the significance before to draw firm conclusions on a potential synergistic effect of their drug. Another improvement would be to assess the anti-PD-1/anti-GDF15 combination in another murine syngeneic model, as the data presented in humanized mice did not address the anti-PD-1/-GDF15 combination. Figure 4, regarding analysis of T-cell infiltration (e.g. in panels f, i, j), the sample size and statistics make it hard to interpret/conclude (2-to-4 points in each condition). It's unclear why the sample size is so small, compared to the number of mice analyzed in the previous figure (10 in each treatment group). These experiments need to be confirmed with at least 6-to-10 mice per group? 7-Figure 4 m-n data: as indicated in the legend, CD45 and CD3 data presented in panel (m) derived from two independent experiments, and in (n), results from the second experiment only are presented to inform on the CD4 and CD8 infiltrates. However, (n) shows data from the control condition derived from 1 mouse. Therefore, it's unclear from where come the measures in (m). For clarification, it would be reasonable to show the distribution of CD4 and CD8 from the two independent experiments. 8-Page13 L20: authors claim "GDF-15 hence represents an independent marker for failure of anti-PD-1 therapy" It is not clear whether the authors have done a multivariate analysis to conclude that GDF-15 separates from other known risk factors. This would be an important point given the poor HR and significant p values presented in Table S2. If so, other variables considered in the model should be presented with HR and p values. 9-Also, have the authors examined the potential association with PD-L1? Or test if GDF-15 is a better biomarker than PD-L1? 10- Figure 6: For clarity, authors should annotate with the name of each cohort and insert the sample size "in each subgroup". Some dots have been removed in some conditions like in panel b, but it is important to know how many patients have been included for statistical analyses. 11-Discussion, p16 and abstract: author claim: "We show that tumor-derived GDF-15 inhibits LFA-1 activation on human cytotoxic T cells, thereby interfering with T cell adhesion, rolling and diapedesis". Using the term "cytotoxic" in this sentence sounds like an overstatement as experiments were not performed to test the cytotoxic features of effector cells (cytolytic markers, cytotoxic activity of CTL or NK cells, etc…). Moreover, as mentioned above, the mechanistic part (GDF15/LFA1) was performed with peripheral T lymphocytes from healthy donors.

6-
12-as shown for the MC38 model (Fig. 4h), it would be useful to provide IHC staining from the humanized model to visualize the CD8 T-cell infiltrates in the isotype and anti-GDF15 conditions.
Minor comments: 13-figure 4 a, h: the quality of the photomicrographs should be improved. Additionally, in h, as in a, authors should indicate what immunostaining was performed.
14-Authors claims no relationship was found between GDF15 expression and tumor mutational burden (supp Figure 8). Since GDF-15 is a downstream target of TP53, it would be interesting to include a comparison of the GDF-15 low/high groups in patients stratified for TP53 status and assess if it could relate to ICB response.
15-page 8 L15: to identify the T-cell subsets affected by GDF-15, purified immune cells were briefly exposed to . If appropriate, authors should specify "isolated from healthy donor PBMC". Figure 3h: is for survival comparison, authors should edit the Y axis legend to make it clear.

16-
17-discussion should be improved: for instance, authors should better highlight what is new in their study with respect to previous reports on the link between GDF-15 and tumor immune escape (ref. 23 and 27). They also need to clearly state the limitations of their study and discuss the characteristic of the cohort (e.g. Pts with Brain metastasis) relative to other types of patients treated with ICB. A discussion about the potential of GDF15 as a biomarker for prognostic or predictive.
18-Method p23 immunohistochemistry: authors should indicate who performed and if the analyses were blinded to the treatment outcomes. Figure 5 legend and P23 method: "Formalin-fixed paraffin-embedded tissues from 70 patients with melanoma brain metastases", it is unclear if the tissues analyzed are from brain metastases or primary tumor tissues obtained from metastatic patients. 20-P1 L3," Having established a link between GDF-15 and T cell migration in mice, we assessed GDF-15 expression": T-cell "infiltration" or "adhesion" seems more appropriate 21-The statement in the title « Tumor-derived GDF-15 acts as roadblock for LFA-1-dependent Tcell recruitment and suppresses response to immune checkpoint inhibition» is entirely proven since the outcome values for anti-CTLA-4 in the studied cohort were not always conclusive. Moreover, anti-CTLA-4 treatment has not been investigated in the MC38 model. Authors should consider revising the title and adjusting stated claims Reviewer #3 (Remarks to the Author):

19-
The manuscript by Haake and colleagues postulates a new mechanism by which GDF-15 contributes to immune evasion by tumor cells, namely suppression of LFA-1 dependent recruitment. They also show the potential clinical significance of this by providing data that suggest GDF-15 suppresses responses evoked by anti-PD-1 therapy. The strengths of this manuscript are its novelty given that investigations into the contribution of GDF-15 into tumor immunology are relatively rare (PMIDs 20534737, 32508832) and the use of syngeneic murine models, humanised mice models and patient samples to confirm these effects. The main weaknesses is the lack of evidence suggesting that the mechanism by which GDF-15 suppresses immune responses in vivo is T cell intrinsic and trafficking dependent, much less that this is LFA-1 specific.
Major comments -The manuscript shows some evidence that GDF-15 affects T cell migration in vitro but there is limited evidence that a) Therapeutic effects in vivo are T cell dependent b) Effects are T cell intrinsic or c) mediated through T cell trafficking. Some evidence is presented pertaining to point c in Figure 4 but this is performed in a very limited cohort size and would need a greater n number to be convincing.
-The authors provide data inferring that MC38 tumors can be further sensitised to anti-PD-1 therapy through blockade of GDF-15. However, MC38 is already quite PD-1 sensitive and therefore it would add further signfance to the paper if results could be replicated in a second syngeneic model that is less sensitive to anti-PD-1 at baseline.
-One point of concern is that there appears to be major inconsistencies in tumor growth between experiments even when the same conditions are tested. For example in Figure 3H approximately 50% of parental MC38 tumors are cleared but in 3i this is 0%. Similarly in Figure 3J MC38tgGDF-15 tumors (vehicle treated) uniformly reach 2000mm3 by approximately day 25 but this appears to be <500mm3 in 3K. I could not see a reason for these differences, can this be explained? -In Figure 5 and 6 the authors present very interesting patient data that indicates patients with elevated GDF-15 have poorer overall survival. Of course with any such dataset one must keep in mind responses may be correlative but not necessarily causative. With this in mind, to what extent do GDF-15 levels correlate with TGFbeta (part of the same family and known to lead to an immune excluded phenotype) and does this explain these observations in patients?
-The authors should discuss why they chose to overexpress human GDF-15 in the mouse model as opposed to murine GDF-15. The immune response mounted against human GDF-15 is clearly a caveat of the current work.

Minor comments
LFA-1 is also important for T cells binding to tumor cells and antigen presenting cells. Did the authors consider/ evaluate whether these parameters are affected by GDF-15? Simple in vitro analyses could be performed e.g. killing assays, conjugate assays to address this.
In terms of the T cell intrinsic nature of the in vivo effects, the authors indicate that the GFRAL receptor is not expressed on lymphocytes but the mechanism by which they are affected is not really discussed. Could the authors elaborate on this topic. Reviewer #1 (Remarks to the Author): In this comprehensive study by Haake and colleagues, the authors address the role of the TGFb family member GDF-15 in suppression of anti-tumor T cell activities and raises a new concept, namely that secretion of this cytokine in the tumor microenvironment (TME) promotes a novel mechanism of tumor evasion at least in some types of tumors. To this end, the authors use elegant sets of in vitro read-outs with nice clinical data from different groups of cancer patients. Overall, this is an interesting study, but I have several reservations, which need to be carefully addressed experimentally or textually.
GDF-15 is a known inhibitor of LFA-1 activation-a classical paper by Kempf (Nature Medicine, 2011) has dissected the molecular basis of this inhibition, especially on neutrophils. The finding that GDF-15 inhibits T cell infiltration via suppression of LFA-1 is therefore not particularly novel. What is intriguing is the final outcome-the apparently poor infiltration and killing functions of CTLs on tumor targets combined with the elevated levels of this cytokine in tumors, but the mechanistic details of these observations are not systematically dissected in the tumor mice models presented in this work.
Major comments 1. CXCL12 is not physiologically relevant for tumor killing T cells-although a nice tool, a CXCL12 based LFA-1 activation readout is insufficient. Matching experiments assessing CXCR3-mediated LFA-1 activation of adhesion (using canonical tumor chemokines such as CXCL9 or CXCL10) will be nice to include.   Figure R1: GDF-15 impairs adhesion of activated T cells to stimulated HUVEC in the presence of CXCL9 and CXCL10. Phase contrast microscopy in chamber slides was used to enumerate T cells that adhere to HUVECs under flow conditions, in the absence or presence of rhGDF-15 or the blocking anti-LFA-1 antibody TS1/18. HUVECs in the presence of  CXCL9 and CXCL10 may appear slightly smaller than the corresponding effect in the  presence of CXCL12, effects of anti LFA-1 were in a similar range as those achieved  with GDF-15 (please see above), and the p value is highly significant (p<0.0001). We thus consider this variability to be donor-and assay-dependent, and abstain from discussing effect sizes in the context of CXCR3 or CXCR4 mediated T cell adhesion. As shown below, CXCL9-and CXCL10-induced CD8 + T cell adhesion to HUVECs can not only be inhibited by . The inhibitory effect of GDF-15 can also be reverted by our anti GDF-15 antibody CTL-002, but not by an isotype control antibody. GDF-15 can thus exert highly significant and specific effects in the presence of different chemokines. As CXCL12 is indeed a nice tool for in vitro assays, and as the generated data were now also reproduced with CXCL9 and CXCL10, and as all these findings are also supported by additional tumor models in vivo, there can be no reasonable doubt that GDF-15 impairs T cell adhesion to endothelial cells.  3. The idea to express human GDF-15 in a murine tumor is elegant, especially for the purposes of direct GDF-15 blocking experiments, but it is critical to repeat these experiments with lines that express the murine analogue and show that its effect resemble those of the human cytokine since after all the responding immune cells in the TME are murine and not human.

Please note: While effects of GDF-15 on T cell adhesion to
Another complication of this model is the induction of anti-human GDF-15 in some of the mice. This introduces noise and complicates the interpretation.
We thank the referee for recognizing the rationale underlying overexpression of   In Figure 3B, MC38 blank and MC38 tghGDF-15 cells were injected in immunodeficient NCI nu/nu -mice, in the former Figure 4d (  6. The in vitro inhibition studies with anti LFA-1 and GDF-15 are convincing but it is essential to perform also combination experiments to further strengthen these data. For instance, in the presence of LFA-1 blocking, does GDF-15 exert any additional inhibitory activity on T effector adhesion? I wouldn't think so but it is absolutely required to demonstrate it.
We thank the referee for this question. As GDF-15 has also been described to reduce agonist-induced activation of β1and β3-integrins on platelets (Rossaint,J.,Vestweber,D. & Zarbock,  7. Elegant dSTORM experiments that aim to quantify LFA-1 activation were performed and presented in Figure 2 but the images are unclear and very hard to follow. The rationale of using this fancy methodology is also unclear-FACs based assays with b2 integrin conformation reporter mAbs are as sensitive and are commonly used to assess interference with chemokine mediated inside-out LFA-1 activation.
We have performed FACS-based assays with the conformation-specific 2-integrin antibody mAb24, and with rhICAM-1-Fc. However, these assays revealed no significant effects of i (Figure 2f,g), the weak effect observed via dSTORM microscopy ( Figure 2h-j), and the strong effect on Talin phosphorylation (Figure 2k, 9. In some of the figures, one of the donors doesn't respond to LFA-1 inhibition or to GDF-15 treatment. This puzzling result is not explained. Either more examples must be provided and if multiple donors share this binary behavior, I'd consider dividing these figures to two subgroups of patient T cells. As is, it is a strange result given that all T cells express LFA-1 and are expected to bind ICAM-1 in vitro with similar efficacy. We presume that the referee is referring to Figure S2. In Figures 1a,b Figure 1C). Of note, these differences were also observed when frozen T cells from the same donor were used, which excludes a donor dependency of the effect.
We thus believe that the differences visible in Figure S2 are more likely due to the vulnerable bioactivity of rhGDF-15 rather than to donor-specific differences. One possible explanation is that GDF-15 is an extremely sticky protein that binds to plastics, and can therefore easily be lost upon storage or pipetting. We have now commented on this possible reason for the observed differences (page 9, lines 12-14  10. There are no discussions on how GDF-15 affects the composition of tumor draining lymph nodes (skin). Adoptive transfer experiments with CFSE labeled CD8 and CD4 populations will allow to track both the homing and initial activation of naïve T cells in TdLNs. Memory polyclonal T cells raised against the tumor in donor mice should be also tested.
We thank the referee for this suggestion. As the network of lymphatic vasculature and lymph nodes responsible for draining the pancreas is extremely complex, with many lymphatic vessels in the interlobular spaces of the pancreas and a not-yet-standardized classification of pancreatic nodes, we could not address this question in the Panc02  11. GDF-15 seem to exert multiple effects on the tumor environment via metabolic changes which are associated with cachexia ( Fig. 3a). The authors don't attempt to link this finding to LFA-1 inhibition or to interference with T cell infiltration. This is puzzling and confusing.
We agree with the referee that GDF-15 is sometimes puzzling and confusing. However, recent data show clearly that immunomodulatory effects of     Figure R5: Dose-dependent binding of wild-type or V87R rhGDF-15 to GFRAL was assessed in a sandwich ELISA with rhGFRAL-Fc used for capture and polyclonal antihuman GDF-15 for detection. Optical density as measured by ELISA reader is indicated. Figure R6: HEK293 cells stably transfected with GFRAL/RET51 were stimulated with wild-type or V87R rhGDF-15. The concentration-dependent increase in Erk1/2 phosphorylation was assessed by homogeneous time-resolved fluorescence (HTRF) with Cisbio ERK phospho-T202/Y204 HTRF kit (#64ERKPEG) according to the manufacturer's instructions.  Thus, tissue-protective, immunomodulatory and metabolic effects of GDF-15 are likely mediated via different receptors. Having seen no signs of cachexia in immunocompetent animals with GDF-15 expressing tumors, we believe that we should not attempt to link different functions of GDF-15 which may, or may not, be related. We have thus added a remark referring to possible metabolic effects of GDF-15 in the tumor microenvironment, but refrained from excessive speculation (page 19, lines 15-17). Based on recent data from the literature, we have also added the statement "Immunomodulatory effects of GDF-15 thus appear to be independent of GFRAL" (page 19, lines 12-15), which is also supported by the most recent literature on J.H.,et al. Colchicine acts selectively in the liver to induce hepatokines that inhibit myeloid cell activation. Nat Metab 3, 513-522 (2021)).

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12. The use of humanized mice in this study is questionable and should be re-considered since it is a very complex experimental system. I don't see what advantages it has over immunocompetent mice since as noted, among other deficiencies APCs in these mice are scarce or abnormal. It is not well explained how the different T cells emerge in this system, and how they distribute in the spleen and blood. I'd consider removing this model and describing it in another paper.  S4-8 (2013).). In their competent hands the system is sufficiently well characterized to generate meaningful data. In fact, Referee 3 has even highlighted this model as a particular strength of the manuscript, which strongly argues against removing it. However, additional experiments performed in syngeneic mice with a surrogate antibody against mouse GDF-15 now complement these data.
13. The clinical data and especially the correlations between elevated serum levels of GDF-15 and survival are nice and statistically significant. Nevertheless, in light of the multiple effects of this cytokine it is hard to directly link these results to the main theme of the paper which is LFA-1 inhibition and attenuation of trafficking and function of tumor killer immune cells in the TME. So is the inclusion of data from pregnant women. It is unclear how these data strengthen the main theme of this work. I think it does the very opposite.
We have now removed the data from pregnant women. We also agree with the referee that GDF-15 exerts multiple effects on the immune system, which we have also discussed. Nevertheless, we respectfully point out that the main topic of the manuscript is the functional relevance of GDF-15 as a new target for cancer immunotherapy. Given the known relevance of CD8 + T cell infiltration for responses to anti PD-1, effects of GDF-15 on LFA-1 on T cells provide a very plausible explanation for the clinical findings. The observed effects of T cell trafficking to tumor-draining lymph nodes, which were assessed on request of the referee, provide a further strong rationale for Figure R8: Stimulated T cells were treated with different preparations of rhGDF-15 and/or antibodies for 20 min before being run in µ-slides over a layer of activated huLEC. T cells adhering under hydrodynamic flow conditions were counted. Three donors were tested.
neutralizing GDF-15 in cancer patients. We also explicitly concede that effects on macrophage polarization or dendritic cell maturation may also contribute to GDF-15dependent immune escape. Nevertheless, a comprehensive evaluation of all possible mechanisms would be an excellent topic for a dedicated issue of a journal. In a single article for Nature Communications we can only focus on findings that appear most salient with regard to novelty and relevance.
Minor comments 1. The terminology 'recruitment' used in the legend of Fig. 1 is confusing. In vitro readouts do not involve recruitment. Please change it. The explanation of these in vitro readouts in the main text should be also improved.
The term recruitment has been replaced by adhesion for all in vitro assays apart from Figure 1i. Figure 1i is part of a sequence depicting different steps in the interaction of T cells with endothelial cells. Here, the terms adhesion (1g), transmigration (1h) and recruitment (1i) (meaning recruitment to the other side of the HUVEC layer) are routinely used. Therefore, we would like to stick to the terminology. However, we hope that all in vitro readouts will now be clear.
2. The title "GDF-15 acts on naïve and memory CD8 + , and on CD4 + memory T cells" must be revisited. As far as I can tell, naïve T cells are less responsive to GDF-15.
We have now performed additional experiments which have added to the previous picture. Initially, we had just observed trends with pan and with naïve CD4 + T cells. Now, the p<0.05 level of significance has also been reached for these subsets. In fact, adhesion of naive CD4 + T cells (p=0.001) and of CD8 + T cells (p<0.0001) to HUVEC are also greatly impaired. Further, we have also observed an effect of GDF-15 on regulatory CD4 + T cells. Therefore, we have now revisited the above-mentioned subtitle and changed it into "GDF-15 broadly affects T cell adhesion to endothelial cells". The question whether specific subsets are more or less responsive may be addressed in the future. Our current insights are accurately represented by the data shown in Figure S2f. Therefore, we would rather confine ourselves to showing the data and concluding that GDF-15 acts broadly on different T cell subsets (which is also supported by preliminary data from our clinical phase 1 trial). Readers who want to infer further details from the data are, of course, free to do so.
3. The mentioning of "catch bonds" is relevant to the discussion but not to the result section since this biophysical property is not directly measured in this study.
The referee is right in pointing out that this biophysical property was not directly measured. Still, readers who are less familiar with integrin signaling will be surprised that minimal differences in m24 and ICAM-1-Fc binding can translate into strongly reduced adhesion. We have now re-arranged and extended Figure 2. By first showing the loss of adhesion to ICAM-1, then presenting the data which indicate that opening of the "headpiece" of LFA-1 is hardly affected by and (2017)), while the very prominent signals were obtained with the anti CD8a antibody. This mistake has now been corrected in the revised Figure (now 3i). In addition, we have slightly adjusted the color/brightness on the complete images to improve the visual impression.
5. Adjuvant immunization of tumor bearing mice is mentioned in the methods but never discussed in the main text. Why?
We apologize for a lack of clarity. The term "adjuvant immunization" referred to immune stimulatory treatment with poly(I:C) and anti-CD40 antibody. However, as the newly added data direct the focus of the manuscript even more on immune infiltration and on the synergy between anti GDF-15 and anti PD-1, we have now omitted the data obtained with (poly(I:C) + anti-CD40) treatment, as these might rather distract the reader. (Moreover, we have also reached the word limit allowed by the journal.) Nevertheless, if the referee feels that these data should still be incorporated, we could also add them again to the manuscript.
6. Why can't alternative adhesive pathways such as VLA-4-VCAM-1 compensate for LFA-1 inhibition in the various models. This point has to be discussed.
Effects of GDF-15 on LFA-1 occur within seconds to minutes, which leaves little room for compensatory mechanisms to be activated. Moreover, expression of integrin ligands differs between different tissues, which limits the potential for compensation through other integrins. Finally, an early study on mice lacking the LFA-1 alpha chain CD11a found that LFA-1-deficient mice did not reject immunogenic tumors grafted into footpads and did not demonstrate priming response against tumor-specific antigen. Med 183, 1415Med 183, -1426Med 183, (1996, new reference 13). A very recent reference also shows that LFA-1 activation enriches tumor-specific T cells in an immunologically "cold" The referee is right that LFA-1 also stabilizes immunological synapses. However, while this is likely true for killing synapses between CTLs and ICAM-1 expressing targets, reference data on www.proteinatlas.org indicate that "most malignant tissues were negative´(for ICAM-1). Rare cases of squamous cell carcinoma, liver cancer and breast cancer showed moderate to strong cytoplasmic and membranous immunoreactivity." Thus, effects on killing synapses will be highly variable. ICAM-1 can, however, greatly stabilize immune synapses between dendritic cells and T cells. This is now also mentioned in page 19, lines 21-22. In this study, Haake and colleagues have investigated the influence of GDF-15 on T-cell recruitment within the tumor microenvironment and on the response to anti-PD-1 immunotherapy. In the first part of the work, the authors provided in vitro evidence of a role of GDF-15 in interfering with the LFA-1/ICAM1 interaction, which is known to be essential for T-cell migration and extravasation. In a second part, using the murine MC38 colon cancer model, they attempted to functionally validate these observations in vivo, and investigated the consequences of GDF15 overexpression on T-cell infiltration and response/resistance to anti-PD1 therapy. In addition, they assessed the potential of using anti-GDF15 to improve anti-PD-1 effect in this setting. Data confirmed the prognostic value of GDF15, and a trend to a synergistic effect for the anti-PD1/anti-GDF15 combination was observed in this model. In the last part, the authors examined various datasets and cohort of patients (Melanoma and oropharyngeal squamous cell carcinomas) to test the impact of GDF15 expression levels (by IHC and serum measures) on patient clinical outcomes and tumor T-cell infiltration. They show a positive association of GDF-15 with poor survival, and inverse correlation with T-cell infiltrates (CD8, CD3, Treg). They also report evidence in two series of metastatic melanoma patients that elevated GDF-15 serum levels are associated with resistance to PD-1 blockade monotherapy, suggesting GDF-15 as a novel predictive biomarker in this cancer type. The topic is of interest and original. The study provides results of relevance for the oncoimmunology field. However, several technical and conceptual concerns need to be addressed.

The observation that these mice mounted normal T cell responses to lymphocyte choriomeningitis virus (LCMV) and vesicular stomatitis virus (VSV) infections and exhibited normal ex vivo CTL function indicates that compensatory mechanisms may exist for some, but not for all functions of LFA-1 (Schmits, R., et al. LFA-1-deficient mice show normal CTL responses to virus but fail to reject immunogenic tumor. J Exp
Major comments: 1-in the first part of the manuscript, in vitro experiments and associated conclusions derive from T cells purified from healthy donor PBMCs. The significance would be stronger if the authors could expand the work and confirm the findings using T lymphocytes from cancer patients. We thank the referee for this suggestion. Unfortunately, our existing ethical approval (15/18-me) does not allow us to obtain sufficient numbers of T cells from cancer patients to perform flow-adhesion assays. Flow cytometry-based ligand capture experiments with hICAM-1-Fc and stainings with m24 anti-human active LFA-1, which can be performed with small blood samples, turned out to be poorly suited to assess responses to GDF-15 -no matter whether cells were obtained from cancer patients or from healthy controls (see Figure 2f,g and also our response to Q7 by referee 1). This is now mentioned (page 8, lines 9-11 Figure S2, authors claim that "GDF-15 acts on naïve and memory CD8+, and on CD4+ memory T cells". But it is unclear how they have differentiated the memory subsets from naïve T cells. Information is missing in the method and Figure. It would be important to provide flow cytometry data to support the observed differences and statements (GDF15/anti-LFA1 treatments). Of note, in figure S2, adhesion data are shown for pan-, naïve-, Memory and EM (effector memory) CD4+ T cells. However, for CD8+ cells, the EM category is missing. Moreover, the quality of the images in panels a and b needs to be improved.
We 3-Data in figure 3 are difficult to interpret. For example, sample size and statistics in panels c, f, g make the current data questionable and hard to interpret (with 2-to-4 points in each group) The data in panels 3c,f and g were meant to show the caveats of our initial mouse model.  Figure 3k and Page 10 (L14), authors claim "Adding anti-GDF-15 to anti-PD-1, however, resulted in complete clearance of MC38hGDF-15 tumors in 4/10 mice (Fig. 3k), indicating that anti-GDF-15 and PD-1-based immune checkpoint blockade synergize to increase survival in mice". This is a key statement but this relies only on 4 events, with no significant differences for survival between the two conditions (p= 0.17). Figure R9: Combined tumor growth curves from the experiment shown in Figure 3t (former Figure 3j).

5-
This could be an interesting observation, but parts of the experiments should be repeated to foster the significance before to draw firm conclusions on a potential synergistic effect of their drug. Another improvement would be to assess the anti-PD-1/anti-GDF15 combination in another murine syngeneic model, as the data presented in humanized mice did not address the anti-PD-1/-GDF15 combination.
We agree that a larger number of mice would be desirable. However, the experiment now shown in Figure 3s and t already included 60 mice for overall survival analysis, and additional mice for the analysis of immune cell infiltration, which was at the limit of feasibility. We further have to accept that German laws for animal protection do not allow for experiments to be repeated.  Figure 4a). The new Figure 4b shows female mice that were inoculated with 10 6

Panc02-luc cells in the tail of the pancreas. Tumor growth was monitored by luciferasebased in vivo bioluminescence imaging. Both experiments showed that monotherapy with anti-PD-1 or anti-GDF-15 is hardly effective in most mice, whereas the combined anti-PD-1 + anti-GDF-15 treatment resulted in tumor clearance in 7/12 or, respectively, 5/12 mice (unadjusted p values: p=0.007 in a) and p=0.0205 in b) as calculated by Mann-Whitney test, adjusted p values p=0.021 in a) and p=0.063 in b), after correction for multiple testing according to Bonferroni-Holm). Thus, we thank the referee for suggesting to assess the anti-PD-1/anti-GDF15 combination in another murine syngeneic model. These new data greatly strengthen our manuscript. Applying the anti-PD-1 + anti-GDF-15 combination to humanized mice would not be promising as these mice largely lack dendritic cells (see page 13, lines 4-7 and reference 45). Based on a personal communication from Sara Colombetti (Roche), Roche only uses humanized mouse models to study immune cell trafficking or in conjunction with agonistic antibodies/biologicals, but no longer with anti PD-L1 or other immune checkpoint blockers.
6- Figure 4, regarding analysis of T-cell infiltration (e.g. in panels f, i, j), the sample size and statistics make it hard to interpret/conclude (2-to-4 points in each condition). It's unclear why the sample size is so small, compared to the number of mice analyzed in the previous figure (10 in each treatment group). These experiments need to be confirmed with at least 6-to-10 mice per group?
Tumor-infiltrating T cells can only be analyzed from tumors that are not cleared by the immune system. Accordingly, analyzing TILs in "non-responder tumors" from experiments with responders would have introduced a massive bias. Thus, additional mice (6 per treatment group) were included to analyze immune cell infiltration at earlier time points. Unfortunately, however, MC38 blank cells were spontaneously rejected in some of these animals, which explains the groups with only 2 animals in the former Figure 4e,f,g (now shifted to Figure 3m- Figure 4 m-n data: as indicated in the legend, CD45 and CD3 data presented in panel (m) derived from two independent experiments, and in (n), results from the second experiment only are presented to inform on the CD4 and CD8 infiltrates. However, (n) shows data from the control condition derived from 1 mouse. Therefore, it's unclear from where come the measures in (m). For clarification, it would be reasonable to show the distribution of CD4 and CD8 from the two independent experiments. Figure 4n was only included in the second experiment. In this experiment, animals Iso1, Iso 3 and Iso 4 showed no T cell infiltrates at all. Apparently, this fact, which led to the absence of bars in these three mice, created the misleading impression that only one mouse had been analyzed (only one bar shown from four mice). We apologize for not having represented the negative T cell infiltration data from these three mice more clearly. This has now been clarified (see revised Figure 4m).

This seems to be a misunderstanding. While CD45 + , CD3 + and CD19 + tumorinfiltrating cells were analyzed in two independent experiments, the T cell subset analysis presented in
8-Page13 L20: authors claim "GDF-15 hence represents an independent marker for failure of anti-PD-1 therapy" It is not clear whether the authors have done a multivariate analysis to conclude that GDF-15 separates from other known risk factors. This would be an important point given the poor HR and significant p values presented in Table S2. If so, other variables considered in the model should be presented with HR and p values.
We have now performed additional multivariate analyses on the Tübingen cohort, where we had additional data on age, sex, line of treatment, absence or presence of brain metastasis, serum LDH and S100B levels. In this analysis, the prognostic relevance of GDF-15 (defined by a cut-off value of 2.0 ng/ml) still yielded a p value of 0.001, and a hazard ratio of 3.312 (95% CI 1.617 -6.783), which was only second to brain metastasis (p<0.001), but superior to LDH (p=0.008). S100B, age, sex and line of treatment were no significant predictors in this analysis. These data are now shown in Supplementary Figure 7 (Table S2). We hope that the additional multi-variate analysis, the actual shape of the curve and the p values may now help the reader to grasp this information more easily.
9-Also, have the authors examined the potential association with PD-L1? Or test if GDF-15 is a better biomarker than PD-L1?
Using the "R2: Genomics Analysis and Visualization Platform (http://r2.amc.nl)", we have performed an in silico correlation analysis to explore whether GDF-15 mRNA expression correlates with PD-L1/CD274 mRNA expression in melanoma. However, none of the available data sets showed a significant positive correlation between GDF-15 and PD-L1. The negative correlation observed in one data set is very weak (R=-0.107, p=0.020) and unlikely to explain the strong clinical impact of GDF-15 levels.  Examples for the stainings (one positive and one negative example) are shown below: Figure R11: Immunohistochemical staining of melanoma sections for MLANA (Melan-A), PD-L1 and S100B.

However, as only 1 out of 14 melanomas was positive for PD-L1, a prediction of response based on PD-L1 would not have been possible in this cohort. Our data thus imply that GDF-15 was a better marker than PD-L1 in this specific cohort.
10- Figure 6: For clarity, authors should annotate with the name of each cohort and insert the sample size "in each subgroup". Some dots have been removed in some conditions like in panel b, but it is important to know how many patients have been included for statistical analyses. Figure 6a. However, no dots have been removed. The only patient who was neglected for statistical analysis was a melanoma patient in Figure 6f, who was initially classified as a complete responder, but then rapidly diagnosed with further metastases. In this case, the discordance between diagnosis and clinical course raised serious doubts regarding the initial classification. Therefore, removal from statistical analysis appeared to be the most appropriate option.

Panel 6b depicts the subset of patients classified as responders to anti-CTLA-4. By definition, this is thus a subgroup of the total anti-CTLA-4 treated cohort shown in
11-Discussion, p16 and abstract: author claim: "We show that tumor-derived GDF-15 inhibits LFA-1 activation on human cytotoxic T cells, thereby interfering with T cell adhesion, rolling and diapedesis". Using the term "cytotoxic" in this sentence sounds like an overstatement as experiments were not performed to test the cytotoxic features of effector cells (cytolytic markers, cytotoxic activity of CTL or NK cells, etc…). Moreover, as mentioned above, the mechanistic part (GDF15/LFA1) was performed with peripheral T lymphocytes from healthy donors.
We thank the referee for this comment. We have now replaced cytotoxic by CD8 + (where appropriate) or just omitted the adjective "cytotoxic".
12-as shown for the MC38 model (Fig. 4h), it would be useful to provide IHC staining from the humanized model to visualize the CD8 T-cell infiltrates in the isotype and anti-GDF15 conditions.
We apologize. As the complete tumors were dissected and analyzed by flow cytometry, there were no tumor samples left for IHC staining.
Minor comments: 13-figure 4 a, h: the quality of the photomicrographs should be improved. Additionally, in h, as in a, authors should indicate what immunostaining was performed.
Regarding the former Figure 4a (now 3i), we had made a mistake and confused rows and columns when we labeled the figure. This has now been corrected. We have also slightly adjusted the color/brightness on the complete images to improve the visual impression. We would also like to point out that myeloid cells give weaker signals than lymphoid cells when stained for CD45. The low number of tumor-infiltrating T cells observed in the MC38 tghGDF-15 tumor may therefore lead to a weaker CD45 signal. With regard to the former Figure 4h (now 3p), we have taken new photomicrographs using a higher magnification. We now provide these more detailed pictures.
14-Authors claims no relationship was found between GDF15 expression and tumor mutational burden (supp Figure 8) 15-page 8 L15: to identify the T-cell subsets affected by GDF-15, purified immune cells were briefly exposed to GDF-15. If appropriate, authors should specify "isolated from healthy donor PBMC".
The wording has been adjusted as suggested by the referee. Figure 3h: is for survival comparison, authors should edit the Y axis legend to make it clear. Figure 3d in the revised manuscript, we have relabeled the Y axis as suggested. For accuracy, we have now indicated in the legend that termination criterion is defined as tumor size > 1200 mm 3 . 17-discussion should be improved: for instance, authors should better highlight what is new in their study with respect to previous reports on the link between GDF-15 and tumor immune escape (ref. 23 and 27). They also need to clearly state the limitations of their study and discuss the characteristic of the cohort (e.g. Pts with Brain metastasis) relative to other types of patients treated with ICB. A discussion about the potential of GDF15 as a biomarker for prognostic or predictive.

In this figure, which has now become
We have done our best to improve the discussion as suggested. We have also clarified that correlations between GDF-15 and T cell infiltration in human tumors were assessed in brain metastases from melanoma, as these lesions are typically only resected once they have grown to a size that allows for a proper histopathological assessment. Other melanoma metastases are, in contrast, either removed as early as possible, or not at all (page 17, lines 14-17). However, the clinical cohorts shown in Figure 6 were not limited to patients with brain metastasis.
18-Method p23 immunohistochemistry: authors should indicate who performed and if the analyses were blinded to the treatment outcomes.
All stainings were performed on the automated IHC staining system Discovery XT and evaluated by an observer who was blinded with regard to the sample or treatment group. This is now indicated (page 28, lines 15-18). Figure 5 legend and P23 method: "Formalin-fixed paraffin-embedded tissues from 70 patients with melanoma brain metastases", it is unclear if the tissues analyzed are from brain metastases or primary tumor tissues obtained from metastatic patients.

19-
The stained tissue specimens were obtained from surgically resected brain metastases. This has now been clarified (page 17, lines 14-17). A challenge when analyzing immune infiltration in melanoma is the fact that easily accessible metastases are resected once they become visible. On the other hand, as surgery is not curative in metastatic melanoma, difficult-to-access lesions are rarely excised. Removal of brain metastases can, in contrast, often improve the quality of life for patients with incurable disease. Therefore, brain metastases are often removed once they have grown a certain size, which makes them highly suited for analyzing T cell infiltration.
20-P1 L3," Having established a link between GDF-15 and T cell migration in mice, we assessed GDF-15 expression": T-cell "infiltration" or "adhesion" seems more appropriate We thank the referee for this suggestion. We have now used the term infiltration.

21-
The statement in the title « Tumor-derived GDF-15 acts as roadblock for LFA-1-dependent T-cell recruitment and suppresses response to immune checkpoint inhibition» is entirely proven since the outcome values for anti-CTLA-4 in the studied cohort were not always conclusive. Moreover, anti-CTLA-4 treatment has not been investigated in the MC38 model. Authors should consider revising the title and adjusting stated claims We have now rephrased the title as « Tumor-derived GDF-15 blocks LFA-1-dependent T-cell recruitment and suppresses response to PD-1-based immune checkpoint inhibition», which is now also in line with the recommended maximum length (15 words) for a title.
The manuscript by Haake and colleagues postulates a new mechanism by which GDF-15 contributes to immune evasion by tumor cells, namely suppression of LFA-1 dependent recruitment. They also show the potential clinical significance of this by providing data that suggest GDF-15 suppresses responses evoked by anti-PD-1 therapy. The strengths of this manuscript are its novelty given that investigations into the contribution of GDF-15 into tumor immunology are relatively rare (PMIDs 20534737, 32508832) and the use of syngeneic murine models, humanised mice models and patient samples to confirm these effects. The main weaknesses is the lack of evidence suggesting that the mechanism by which GDF-15 suppresses immune responses in vivo is T cell intrinsic and trafficking dependent, much less that this is LFA-1 specific.
Major comments -The manuscript shows some evidence that GDF-15 affects T cell migration in vitro but there is limited evidence that a) Therapeutic effects in vivo are T cell dependent b) Effects are T cell intrinsic or c) mediated through T cell trafficking. Some evidence is presented pertaining to point c in Figure 4 but this is performed in a very limited cohort size and would need a greater n number to be convincing -The authors provide data inferring that MC38 tumors can be further sensitized to anti-PD-1 therapy through blockade of GDF-15. However, MC38 is already quite PD-1 sensitive and therefore it would add further significance to the paper if results could be replicated in a second syngeneic model that is hardly sensitive to anti-PD-1 at baseline. Figure 3s and Supplementary Figure 4c). In fact, as wild-type MC38 cells express hardly any GDF-15, blocking GDF-15 would make little sense with these cells. However, we then show that overexpression of hGDF-15 renders the MC38 model almost completely resistant to anti PD-1 treatment, which can be (partly) reversed with an anti GDF-15 antibody (Figure 3t and Supplementary Figure 4d). Using an intrinsically PD-1 resistant tumor model would not have enabled us to show that ectopically expressed GDF-15 can protect a tumor against anti PD-1 treatment. However, while murine tumors generally express much less GDF-15 than human ones, we have now generated additional data to show that the effect of anti PD-1 treatment is synergistically enhanced by anti  in the hard-to-treat orthotopic Panc02 tumor model (new Figure 4a,b). So we now show that a PD-1-sensitive, GDF-15 low tumor can be rendered PD-1 resistant by overexpressing GDF-15, which can be reverted by blocking GDF-15, and we also show that a PD-1 resistant, GDF-15 high tumor can be sensitized to anti-PD-1 treatment by blocking GDF-15.

The referee is right that the MC38 model is highly sensitive to anti PD-1. This is also confirmed by our experiment with mock-transfected MC38 cells (now
-One point of concern is that there appears to be major inconsistencies in tumor growth between experiments even when the same conditions are tested. For example in Figure 3H approximately 50% of parental MC38 tumors are cleared but in 3i this is 0%. Similarly in Figure 3J MC38tgGDF-15 tumors (vehicle treated) uniformly reach 2000mm3 by approximately day 25 but this appears to be <500mm3 in 3K. I could not see a reason for these differences, can this be explained?
We apologize for any potential misunderstanding, but we have not observed 50% tumor clearance in the former Figure 3h (which has now become 3d). We have just observed a significantly slower tumor growth of MC38 blank compared to MC38 tghGDF-15 tumors in immunocompetent mice, with a 100% tumor take rate in this specific experiment (please compare tumor growth curves in Supplementary Figure 4b). Strikingly, these sublines showed an opposite growth behavior in immunodeficient mice. Still, the referee is right that growth rates differed between different experiments. As these experiments were performed by a contracted CRO (Charles River US), we cannot tell whether these differences were linked to differences regarding the fitness of cells, the person performing the experiment, or to microbiota, where minor changes in the animal facility can have strong effects in immunocompetent tumor models. However, as we have always included the relevant controls in the same experiment, we believe that this biological variation does not affect the validity of our data. Seeing that GDF-15 dependent effects persist despite certain differences between individual experiments rather indicates to us that the effects are robust.
-In Figure 5 and 6 the authors present very interesting patient data that indicates patients with elevated GDF-15 have poorer overall survival. Of course with any such dataset one must keep in mind responses may be correlative but not necessarily causative. With this in mind, to what extent do GDF-15 levels correlate with TGFbeta (part of the same family and known to lead to an immune excluded phenotype) and does this explain these observations in patients?
We thank the referee for this valuable remark. There is indeed a list of publications that wrongly assigned effects of TGF- to     -The authors should discuss why they chose to overexpress human GDF-15 in the mouse model as opposed to murine GDF-15. The immune response mounted against human GDF-15 is clearly a caveat of the current work.

excludes T cells and correlates with failure of anti PD-1 therapy would be caused by an (overlooked) association between GDF-15 and TGF- expression. This is now discussed (page 17, lines 11-14) and a key reference describing TGF--mediated T cell exclusion is now cited (Tauriello
While we had to recognize that the immune response mounted against human GDF-15 is indeed a caveat, the choice of model enabled us to use our own antibody, which has now entered a phase 2 clinical trial, to block GDF-15. From a translational perspective, this was an important advantage. Moreover, the strong immunosuppressive effect of human

Minor comments
LFA-1 is also important for T cells binding to tumor cells and antigen presenting cells. Did the authors consider/ evaluate whether these parameters are affected by GDF-15? Simple in vitro analyses could be performed e.g. killing assays, conjugate assays to address this. 8, e78618 (2013), reference 24, Zhou et al. 2013, and Zhang, Y., et al. GDF15 Regulates Malat-1 Circular RNA and Inactivates NFkappaB Signaling Leading to Immune Tolerogenic DCs for Preventing Alloimmune Rejection in Heart Transplantation. Front Immunol 9, 2407(2018, reference 26), and we are also working to figure out the precise role of GDF-15 on LFA-1 activity in antigen-presenting cells. However, simple in vitro assays have already been addressed in the aforementioned manuscripts, while a detailed consideration of the effect of GDF-15 in antigen-presenting cells is a complex issue that deserves consideration in a separate manuscript.

Effects of GDF-15 on antigen-presenting cells have already been demonstrated by others (Zhou, Z., et al. Growth differentiation factor-15 suppresses maturation and function of dendritic cells and inhibits tumor-specific immune response. PloS one
In terms of the T cell intrinsic nature of the in vivo effects, the authors indicate that the GFRAL receptor is not expressed on lymphocytes but the mechanism by which they are affected is not really discussed. Could the authors elaborate on this topic. The authors made a very elaborated effort and extensively revised the MS. The textual revisions are very helpful in particular the discussion points that the effects of GDF-15 on anti cancer immunity are beyond its direct role on inhibition of LFA-1 adhesiveness. As for specific experiments I asked for-the combinatorial in vitro inhibition studies with anti LFA-1 and GDF-15 are convincing and the explanations on the use of human GDF-15 in murine systems are helpful. Some of the figures addressed to the reviewers (and listed as R1-R13) should be incorporated as supplementary figures available to the readers. In particular I would strengthen the new parts that implicate GDF-15 as a multi-functional suppressor of anti-tumor immunity beyond its role in lymphocyte trafficking (see please Figures R3, R8). I would also recommend a slight change in the title (responses instead of response).

Minor:
In the discussion, the authors suggest that interference with the LFA-1-ICAM-1 axis inhibits rolling. This is wrong, at least for T cells. LFA-1-ICAM-1 is mainly involved in post rolling arrest, crawling and diapedesis across activated blood vessels.
Reviewer #2 (Remarks to the Author): The authors have adequately responded to most of my comments. However, they were unable to address some concerns for technical reasons or limited access to biological samples. Nevertheless, they should: Major concerns 1-Discuss why flow cytometry failed to show an effect of GDF-15 on the binding of the conformation-specific anti-active-LFA-1 antibody mAb24 (Figure 2f) or an Fc-tagged ICAM-1 complex ( Figure 2g) to CD8+ T cells from healthy donors or cancer patients.
2-Perform additional experiments using positive selection of T lymphocytes with anti-CD8 or anti-CD4 (not anti-CD3) antibodies to limit interference on the results.
3-The authors should be more rigorous in setting their mouse model and interpreting their results. They should also discuss the origin of heterogeneity in anti-human GDF-15 antibodies from one mouse to another.

4-OK
No need to include the data with poly(I:C) + anti-CD40 treatment.
5-OK with the Cox model and the additional Panc02 pancreatic cancer model.
6-Mice that spontaneously reject MC-38 tumors should not be recorded, therefore larger groups of mice need to be included. Moreover, the authors have to provide a more convincing interpretation of the observed results. The explanation that "the injected cells had been rested on ice for too long" is not acceptable. Similarly, "… safely assumed that the number of infiltrating T cells in eradicated tumors was at least as high as in non-eradicated ones" is not acceptable.
7-Again, the number of experiments and number of mice in each experiment is too limited to obtain strong and statistically convincing results.
8-OK with the multivariate analysis.
9-OK with Supplementary Fig. 5 and integration of the negative, but informative, correlation.
10-The authors should be more precise with the patient subsets and explain why they removed a particular patient for statistical analysis reason.

11-OK with CD8 T cells.
12-IHC staining could have been performed on tumors from additional mice, not necessarily from the same groups of mice than FACS.
Minor comments: 13-OK with the correction of the mistake in Fig. 3i and with the new photomicrographs in Fig. 3p.
14-Authors may briefly discuss these statements in the discussion section.
15-OK with the wording.
17-OK with the new version of the discussion, which could be completed as indicated above.
18-OK with immunohistochemistry staining and analysis.

20-OK with T-cell infiltration.
21-OK with the new title.
Reviewer #3 (Remarks to the Author): I thank the authors for addressing all questions, the manuscript is now significantly improved.

Reviewer #1 (Remarks to the Author):
The authors made a very elaborated effort and extensively revised the MS. The textual revisions are very helpful in particular the discussion points that the effects of GDF-15 on anti cancer immunity are beyond its direct role on inhibition of LFA-1 adhesiveness.
As for specific experiments I asked for-the combinatorial in vitro inhibition studies with anti LFA-1 and GDF-15 are convincing and the explanations on the use of human GDF-15 in murine systems are helpful. Some of the figures addressed to the reviewers (and listed as R1-R13) should be incorporated as supplementary figures available to the readers. In particular I would strengthen the new parts that implicate GDF-15 as a multi-functional suppressor of anti-tumor immunity beyond its role in lymphocyte trafficking (see please Figures R3, R8). I would also recommend a slight change in the title (responses instead of response).
We thank the referee for these thoughtful and positive comments. We have now incorporated Figure R8 into Figure 1 (as new Figure 1f). Figure R3 has been incorporated as Supplementary  Figure 5c. Also the title has been changed as proposed.

Minor:
In the discussion, the authors suggest that interference with the LFA-1-ICAM-1 axis inhibits rolling. This is wrong, at least for T cells.
This has been corrected. The sentence (page 17, lines 3-6) now reads "Here we show that GDF-15 also prevents a stable, Talin-dependent linkage between LFA-1 and the actin cytoskeleton. Tumor-derived GDF-15 thus inhibits the LFA 1:ICAM 1 axis in human T cells, thereby interfering with T cell post rolling arrest, firm adhesion to endothelia, crawling and diapedesis across activated blood vessels."

Reviewer #2 (Remarks to the Author):
The authors have adequately responded to most of my comments. However, they were unable to address some concerns for technical reasons or limited access to biological samples. Nevertheless, they should: Major concerns 1-Discuss why flow cytometry failed to show an effect of GDF-15 on the binding of the conformationspecific anti-active-LFA-1 antibody mAb24 (Figure 2f) or an Fc-tagged ICAM-1 complex (Figure 2g) Figure 2h. Thereby, we have now fulfilled all requests raised during the initial review * .
The new request to also use positively selected cells for flow adhesion experiments entails a caveat. Using either human CD8 + micro beads  for positive selection, or the human CD8 + T cells selection kit  for negative selection, CD8 + T cell purity (91% with #130-045-201, 93% with #130-096-495) and the proportion of CD45RO + memory CD8 + T cells purity (37% with #130-045-201, 40% with #130-096-495) were very similar. Despite small differences with LFA-1 blockade, adhesion data were also quite similar for negatively and for positively selected CD8 + T cells. However, beads that remain attached to positively selected cells largely abrogate transmigration. Given that our manuscript explores effects of GDF-15 on T cell infiltration into a tumor tissue, which requires adhesion and transmigration, we believe that we should stick with negatively selected cells.

# T Cells / mm sq.
We would further like to point out that this supplementary figure is only meant to show that GDF-15 broadly affects the adhesion properties of T cell subsets. A differential analysis of GDF-15 effects on individual cell types is an interesting question that could be addressed in a future manuscript. * Original comment by Referee 2: "page 8 and Supp. Figure S2, authors claim that "GDF-15 acts on naïve and memory CD8+, and on CD4+ memory T cells". But it is unclear how they have differentiated the memory subsets from naïve T cells. Information is missing in the method and Figure. It would be important to provide flow cytometry data to support the observed differences and statements (GDF15/anti-LFA1 treatments). Of note, in figure S2, adhesion data are shown for pan-, naïve-, Memory and EM (effector memory) CD4+ T cells. However, for CD8+ cells, the EM category is missing. Moreover, the quality of the images in panels a and b needs to be improved." Figure R2: Negatively or positively isolated human CD8 + T cells were run over a layer of activated human human umbilical vein endothelial cells (HUVEC). T cells adhering (left) and transmigrating (right) under hydrodynamic flow conditions were quantified under a fluorescent microscope.
3-The authors should be more rigorous in setting their mouse model and interpreting their results. They should also discuss the origin of heterogeneity in anti-human GDF-15 antibodies from one mouse to another.
While rigor in designing and interpreting results is indeed key to successful preclinical research, we proudly point out that our preclinical work has been translated into phase 1 and phase 2 clinical trials in human cancer patients. Based on our data we predicted that GDF-15 neutralization can enhance current aPD-1 therapies. This was confirmed in patient biopsies by increased intratumoral T cell infiltration, and by some impressive clinical responses in heavily pre-treated, aPD-1-refractory last-line patients with solid tumors. Please find a brief summary of early clinical trial data in https: //www.catalym.com/wp-content/uploads/2022/09/220910 _ESMO_CTL-002-001-Phase-1-Trial-presentation_MELERO_v1.0.pdf While we cannot include the data generated by a large group of clinical investigators to the present pre-clinical manuscript (which is already filled with data), there can be no better validation of pre-clinical work than its successful translation into patients.
Moreover, protection of animals enjoys legal and constitutional status in Germany, and regulations are becoming continuously stricter. We are only allowed to use animals for research when there is no other way of obtaining the desired result. Once a question has been addressed in an animal experiment, a permission to repeat this experiment will not be granted again. Instead, authorities expect investigators to draw all conclusions from the already available animal data. We cannot ignore and overcome this legislation, which is clearly not in favor of science. Accordingly, we sometimes have to accept that unanticipated problems like poor engraftment of tumor cells in a group of mice may result in less-than-perfect results. Therefore, we have now written (page 10, line 24 to page 11, line 2) "While the number of tumors that could be harvested and assessed by flow cytometry after day 23 was too small to allow for valid conclusions, effects on immune cell infiltration still showed a trend (Figure 3m-o)." We believe that this is an apt description of these admittedly less-than-ideal data. Still, with highly concordant data from four different animal models (transgenic sub-cutaneous MC38 colon cancer, orthotopic Panc02 pancreatic cancer, orthotopic EMT6 breast cancer, and humanized patient-derived xenograft models), detailed mechanistic data and early clinical data that all confirm our hypothesis, the conclusions drawn in the manuscript are extremely well substantiated.
Regarding the heterogeneous development of endogenous antibodies, we have now written on page 10, lines 15-17: "Still, immunosuppressive effects of human GDF-15 likely prevented antibody formation in 50% of the mice, and immunosuppressive effects of human GDF-15 more than outweighed its immunogenicity in mice."

4-OK
No need to include the data with poly(I:C) + anti-CD40 treatment.

Thank you.
5-OK with the Cox model and the additional Panc02 pancreatic cancer model.

Thank you.
6-Mice that spontaneously reject MC-38 tumors should not be recorded, therefore larger groups of mice need to be included. Moreover, the authors have to provide a more convincing interpretation of the observed results. The explanation that "the injected cells had been rested on ice for too long" is not acceptable. Similarly, "… safely assumed that the number of infiltrating T cells in eradicated tumors was at least as high as in non-eradicated ones" is not acceptable.
While we agree with the referee that mice should not be recorded when a tumor does not engraft, we urge the referee to take a closer look at Supplementary Figure 4d: Here, it is clearly visible that several tumors were rejected after engraftment. Excluding these animals would constitute a selection on non-responders and thereby introduce a bias. As rejection of MC38 tumors depends on tumor-infiltrating T cells, it was never observed in T cell deficient mice. We thus maintain that such tumors were evidently infiltrated by T cells. Ignoring these animals would therefore introduce a substantial bias.
Still, the referee is absolutely right that the small number of mice in some groups is clearly suboptimal, and we have also acknowledged this in the manuscript. Page 10, line 24 to page 11, line 2: "While the number of mice that could be assessed by flow cytometry after day 23 was too small to allow for actual conclusions, effects on immune cell infiltration still showed a trend (Figure 3m-o)." However, as outlined above we are not free to perform animal experiments whenever we consider them useful. We have to accept the limitations imposed by law, which does not allow for repeat experiments. We therefore also discussed with the governmental review board of the State of Bayern, Regierung von Unterfranken. Still, from these discussions it became clear that repeating the whole set of animals with MC38 tghGDF-15 tumors will not be approved. Scientifically, however, an addition of individual animals to selected groups is no valid option. As immune responses depend on the microbiome and on the condition of the mouse facility, only littermates must be compared within one experiment. Therefore, we cannot optimize in a way that would be scientifically valid and compatible with the rules for animal experimentation. Figures 4c-e, Figures 4f,g, Figure 4l) and supported by correlations obtained using human tumor tissues ( Figure 5). Data from humans treated with a neutralizing anti-GDF-15 antibody has been presented at ESMO2022 (not shown in this manuscript). The ability of GDF-15 to exclude T cells from the tumor microenvironment is thus very well supported by data. Accordingly, GDF-15 overexpression protects MC38 tumors from anti-PD-1-mediated clearance. Again, the effect of GDF-15 can be reverted by a neutralizing anti-GDF-15 antibody. All these effects are statistically significant and relevant. However, the confounding factor that has shown up in these experiments, namely the development of endogenous antibodies against human GDF-15 in some mice, would likely be observed again if the same experiment was repeated and expanded. Therefore (and due to the legal framework for animal experimentation), we had to find a work-around. Initially, we circumvented the problem by using a humanized patient-derived xenograft model. In the course of the first revision, we then performed additional experiments in an orthotopic Panc02 pancreatic cancer and an orthotopic EMT6 breast cancer model -and obtained concordant results in all four models. In our opinion, showing that GDF-15 acts as a "T cell repellant" in several different models is even more convincing than demonstrating the effect in a maximally optimized model. Importantly, the other two referees who also felt the need for additional animal data, have accepted our strategy as a valid approach to support our hypothesis.

Still, the data we obtained from the transgenic model show unambiguously that expression of GDF-15 confers a survival advantage to tumor cells grown in immune competent, but not in immunodeficient animals. The model further shows that GDF-15 reduces CD8 + T cell infiltration in tumors, which can be reverted by a neutralizing anti-GDF-15 antibody (which also shows that the antibody works in vivo). This effect on T cell infiltration was subsequently confirmed in other models (see
7-Again, the number of experiments and number of mice in each experiment is too limited to obtain strong and statistically convincing results.
In the first response to the referees, we had already outlined that the referee´s perception of the animal number was wrong. We apologize if we have not been clear enough in indicating that several mice treated with the isotype antibody showed no detectable tumor-infiltrating human T cells at all and were therefore difficult to display. Moreover, statistical analysis of tumor-infiltrating immune cells (Figure 4l, unchanged, based on 9 mice per group) yielded p values of 0.029 for CD45 + and 0.012 for CD3 + T cells. B cells, which are less dependent on LFA-1 for trafficking, were assessed as a negative control (p=0.718). At the p<0.05 level of significance, the results for CD45 + and CD3 + cells are statistically convincing. The lack of impact on B cell infiltration was expected. We hope this clarification solves the issue. By calculating the overall percentage of CD4 + , CD8 + , CD4 + CD8 + double-positive and CD4 -CD8double-negative T cells in the disseminated tumors, we have now also been able to perform additional statistics. While no clear data were obtained for double-positive or double-negative T cells (p values 0.20 and 0.62, respectively), treatment with anti-GDF-15 antibody resulted in a statistically significant enrichment of tumor-infiltrating CD4 + T cells (p=0.037). For CD8 + T cells, the level of significance was missed by a narrow margin (p=0.069,. However, as CD8 + T cells were found in 4/4 anti-GDF-15-treated, but only in 1/4 isotype antibody-treated tumors, these results clearly support the findings from other models. Additional animal experiments would thus not be justified. Moreover, humanization of mice based on the chosen protocol exceeds the time foreseen for this revision. Finally, we refer once again to the clinical data obtained with GDF-15 blockade in last-line patients (https://www.catalym.com/wp-content/uploads/2022/09/220910_ESMO_CTL-002-001-Phase-1-Trial-presentation_MELERO_v1.0.pdf). We hope that we will soon be able to share this additional validation with the broader scientific community.

8-OK with the multivariate analysis.
Thank you. Supplementary Fig. 5 and integration of the negative, but informative, correlation.

9-OK with
Thank you.
10-The authors should be more precise with the patient subsets and explain why they removed a particular patient for statistical analysis reason.
One single patient was removed from the statistical comparison of GDF-15 serum levels between different response groups (Figures 6f,g,h). The reason were discordant data between clinical staging and clinical course. Though this patient was staged as a complete responder, he developed symptomatic metastases within weeks after the staging and died from melanoma soon thereafter -which strongly suggests that his initial response was less than complete. Unfortunately, we cannot retrospectively resolve this discrepancy. Therefore, we cannot confidently assign this patient to a response group. (CR would most likely be wrong, though compatible with the staging result. Any other assignment would signify a retrospective change of the staging result, which would not be correct, either.) In the Figure legend we have now written "In f,g,h, one patient whose clinical course contradicted the classification as complete responder was omitted from statistical consideration" (previous version "disregarding one misidentified complete responder", page 52, lines 4-5). In the Materials and Methods part, section on statistics, we have now added "Due to a discordance between staging and clinical course, one patient was neglected in the statistical analyses for Figures 6f,g,h" (page 36,. In the extended Figure legends we had already written: "One patient who relapsed and died from melanoma despite being classified as complete responder was treated as on outlier in the CR group and thus disregarded" (page 71, lines 15-17). We have now added the information that this patient died within months from melanoma.
Still, we thank the referee for directing our attention again towards this detail, as excluding this patient only from statistical analysis in Figure 6f was inconsistent. As the analyses in Figures 6g and h are also based on clinical staging, we have now also excluded this patient from these analyses. In the corresponding statistics odds ratios were thus reduced from 0.796 to 0.778 in Figure 6g and from 0.782 to 0.773 in Figure 6h, while p values changed from 0.0347 to 0.0315 in Figure 6g and from 0.00906 to 0.0085 in Figure 6h. Thus, results are hardly affected by our decision -which is not surprising as just one of 88 patients in this cohort, and none of the 35 patients in our second cohort showed such discordant data. Still, we are convinced that this is the best way of dealing with the contradictory data, and we are now consistent in excluding the questionable staging result. As we would not exclude a patient without good reason, we have still included this patient in the analysis of overall survival, where data were unambiguous.

11-OK with CD8 T cells.
Thank you.
12-IHC staining could have been performed on tumors from additional mice, not necessarily from the same groups of mice than FACS.
As the absolute number of tumor-infiltrating CD8 + T cells is quite low in this humanized mouse model, immunohistochemical analyses suffer from a substantial sampling bias. While we had stained some sections when setting up the model, flow cytometry-based analysis of dissected tumors yields much more representative and therefore also more reliable data. Thus, we have now written on page 13, lines 10-11: "Due to the overall low TIL numbers and the resulting sampling bias, immunohistochemical analyses were inferior to flow cytometry-based infiltration assessments." Minor comments: 13-OK with the correction of the mistake in Fig. 3i and with the new photomicrographs in Fig. 3p.

Thank you.
14-Authors may briefly discuss these statements in the discussion section.

Thank you.
15-OK with the wording.

Thank you.
17-OK with the new version of the discussion, which could be completed as indicated above.

Thank you.
18-OK with immunohistochemistry staining and analysis.

Thank you.
19-OK with FFPE brain metastasis samples.